Genetic engineering broadly, and gene therapy studies, in particular, involve the genetic transformation of living host cells by introduction of exogenous materials such as foreign DNA to the host cells to change or modulate the host cells. When successful, the genes carried by the foreign DNA are expressed in the host cells, and thus, the host cells are transformed. Genetically transformed cells or tissues are of great value in research, medicine and agriculture.
There are several conventional gene transfer techniques which are routinely used such as cell fusion, electroporation, liposome fusion, calcium phosphate precipitation, viral infection, conjugal transfer, micropipette microinjection and aerosol beam microinjection. While much research is dedicated to gene transfer techniques, all of these conventional transfer technologies deal with DNA, DNA/liposome or DNA/protein particles in large, noncondensed forms. Noncondensed DNA particles are less stable since there are nucleases in blood which can destroy noncondensed DNA, and noncondensed DNA particles are likely too large to fit into a cellular endosome following receptor-mediated endocytosis, or to be transported into the nucleus of post-mitotic cells. Therefore, the effective transfer of genes remains an obstacle in many fields of research.
It has been recognized that DNA particles in condensed form are more effective in successfully delivering the DNA payload to target tissues while remaining stable and permitting receptor-mediated endocytosis. With this recognition, the literature teaches adding DNA and condensing proteins together while mixing, generally at low concentrations of each. This produces condensed DNA particles called .psi.-form DNA which consists of approximately 10-50 molecules of DNA and many molecules of condensing proteins. A laboratory method for the production of .psi.-DNA is described in Perales, et al., Biochemical and Functional Characterization of DNA Complexes Capable of Targeting Genes to Hepatocytes via the Asialoglycoprotein Receptor, J. of Biological Chemistry, vol. 272, pp.7398-7407, which is herein incorporated by reference. These .psi.-DNA particles have a size on the order of 100 to 200 nm which are improved over noncondensed particles, but do not completely eliminate the stability, efficiency and specificity problems of the larger particles altogether.
Condensation of DNA particles to smaller sizes than .psi.-DNA has been heretofore achieved manually by highly trained individuals in a lab. A preferred form of DNA particles which are condensed even smaller than the .psi.-DNA is unimolecular, which refers to DNA complexes which consist of a single unit of DNA plasmid; these particles are referred to as compacted DNA particles to distinguish them from condensed DNA particles which consist more typically of multiple molecules of DNA. Due to the multiple steps of such a manual procedure and the number of variables inherent in the operation, the successful production of compacted DNA particles is more akin to an art form practiced by a few qualified individuals rather than a repeatable pharmaceutical production method. These unimolecular formulations of compacted DNA are stable, and, compared to other formulations, more effectively transfer DNA to the nucleus of target tissues following intravenous and other routes of administration.
One of the limitations of conventional gene transfer techniques has been the size of DNA particles which are introduced to target tissues. The instability of large DNA particles and the unsatisfactory level of gene transfer with conventional condensed DNA particles have prompted the use of smaller, compacted forms of DNA particles. A discussion of compacted nucleic acids and their delivery to cells as well as the advantages of using compacted DNA is disclosed in commonly assigned U.S. Pat. Nos. 5,844,107 and 5,877,302, the entire disclosures of which are hereby incorporated by reference. Since the production of compacted DNA manually is expensive, time-consuming and sometimes inconsistent, there is a need for an automated device which produces compacted, unimolecular DNA particles in a controlled, pharmaceutical production method.